I. Field of the Invention
The present invention relates to the fields of molecular and cell biology generally, and more specifically, it addresses mechanisms for growth control in eurkaryotic cells. In particular, there are provided viral genes that inhibit viral protein synthesis and methods for use thereof.
II. Related Art
Herpes simplex viruses, designated with subtypes 1 and 2, are enveloped viruses that are among the most common infectious agents encountered by humans, infecting millions of human subjects worldwide. These viruses cause a broad spectrum of disease which ranges from relatively insignificant to severe and life-threatening. Clinical outcome of herpes infections is dependent upon early diagnosis and prompt initiation of antiviral therapy. Despite some successful efforts in treating HSV infectious, dermal and epidermal lesion often recur, and HSV infections of neonates and infections of the brain are associated with high morbidity and mortality.
Herpes simplex virus 1 (HSV-1) causes two kinds of infection. The first, exemplified most dramatically after first exposure to the virus, results in productive infection at the portal of entry of the virus into the body. In productive infection, approximately 80 different genes are expressed, viral protein and DNA are made, viral progeny is assembled and, ultimately, the cell is destroyed. The second type of infection, latent infection, takes place only in sensory neurons populated by viruses brought to that sites by retrograde transport along axons from the portal of entry. In latently infected cells, viral DNA is maintained as an episome, and the only products detected to date are transcripts arising from two copies of a 8.5 kB domain of the DNA. Recombinants lacking sequences encoding the promoters and 5xe2x80x2 domains of these RNAs, however, are capable of establishing latent infections. These finding suggested that either as yet unknown viral gene products or cellular gene products are responsible for the establishment of the latent state (Roizman and Sears, 1995).
The genes expressed during the productive infection encode numerous functions related not only to viral replication, but also to the increase of the pool of susceptible cells, to more efficient spread of infection from cell to cell and to the abrogation of host response to infection (Ward and Roizman, 1994). In light of these specialized activities, it is rather unlikely that establishment of latency, a vital function for the perpetuation of the virus in human populations, would solely rely on functions expressed by the host cell. Experiments designed to look for viral genes expressed during latency led to the discovery that an open reading frame, designated as ORF P, is repressed during productive infection, but is expressed under conditions where the repressor is not made or the site of binding of the repressor is ablated (Lagunoff and Roizman, 1994; 1995). A definite functional link between this gene product and latency has not been established.
It is, therefore, an object of the present invention to provide methods for the treatment of HSV infections, and compositions therefor. More specifically, it is an object of the present invention to provide methods that inhibit, induce or maintain latency of herpesvirus infections. It also is an object of the present invention to provide methods for the identification of compositions that will inhibit, induce or maintain latency of HSV infections.
In satisfying these objectives, there is provided, in a first embodiment, a method for inducing latency in a herpesvirus infected cell comprising the step of increasing the level of ORF P polypeptide in the cell. The herpesvirus may be a herpes simplex virus and, more particularly, a type 1 herpes simplex virus or a type II herpes simplex virus. The cell may be a human cell and, in a preferred embodiment, a human cell located in a human subject.
Increasing the level of ORF P may comprise providing an ORF P polypeptide to the cell, for example, by contacting the cell with an ORF P polypeptide. Alternatively, providing may comprise contacting the cell with a nucleic acid encoding an ORF P polypeptide. Preferably, the nucleic acid is under the transcriptional control of a promoter active in eukaryotic cells, for example, a herpesvirus promoter. The nucleic acid may further be contained in a replication-deficient vector, such as a replication-deficient herpesvirus vector. The herpesvirus vector may be contained in an infectious herpesvirus particle, where contacting is performed under conditions that permit infection of the cell by the herpesvirus particle. This method may further comprise a step of administering an anti-viral agent to the cell.
In another embodiment, there is provided a method for preventing latency in a herpesvirus infected cell comprising the step of decreasing the level of ORF P polypeptide in the cell. The herpesvirus may be a herpes simplex virus and, more particularly, a type 1 herpes simplex virus or a type II herpes simplex virus. The cell may be a human cell and, in a preferred embodiment, a human cell located in a human subject. Decreasing ORF P may comprise providing to the cell an antisense construct, wherein the antisense construct inhibits transcription or translation of an ORF P gene. The antisense construct may comprise an oligonucleotide that targets a 5xe2x80x2-untranslated region or a translational start site for ORF P. Alternatively, decreasing may comprise inhibiting translational processing of the ORF P polypeptide. In another alternative, decreasing may comprise providing to the cell an antibody that binds immunologically to the ORF P polypeptide. Preferably, the antibody is a monoclonal antibody.
In still another embodiment, there is provided a method for inhibiting the splicing of a RNA transcript comprising the step of contacting the transcript with an ORF P polypeptide. The RNA transcript may be produced in an in vitro transcription system or it may be produced in a cell. The ORF P is produced recombinantly and it may be produced in the same cell as the RNA transcript. The recombinant ORF P may be under the control of an inducible promoter.
In still yet another embodiment, there is provided a method for isolating a splicing factor comprising the steps of (a) providing an ORF P polypeptide; (b) contacting the ORF P polypeptide with a cell extract containing a splicing factor; and (c) isolating the ORF P polypeptide-splicing factor complex. Preferably, the ORF P polypeptide is bound to a support.
In still yet a further embodiment, there is provided a method for isolating an SM antigen comprising the steps of (a) providing an ORF P polypeptide; (b) contacting the ORF P polypeptide with a cell extract containing an SM antigen; and (c) isolating the ORF P polypeptide-SM antigen complex. Preferably, the ORF P polypeptide is bound to a support
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.